It is now well established in the T cell development literature that two key signals, presented in the thymic niche, in a highly controlled and sequential manner, play crucial roles in generating functional T cells: (i) Delta like ligands - Notch receptor signaling and (ii) Major Histocompatibility Complex (MHC) - T cell receptor (TcR) signaling. The Delta like ligands that control Notch signaling and the MHC molecules that direct TcR signaling in the thymus are present on the surface of stromal cells and antigen presenting cells, with which the developing stem/progenitor cells directly interact. It has been shown that at least the Notch signaling process can be mimicked in vitro using retrovirally transfected stromal cells displaying Notch ligands. Recently, we have reported that microbeads with surface-immobilized Notch ligands (delta-like ligand 4, DLL4) can efficiently direct hematopoietic progenitor cells to the early T cell lineage. In addition, we have now demonstrated that, using a combination of surface-immobilized Notch ligand-mediated signaling and exogenous, soluble antigen-loaded MHC tetramer-mediated TcR signaling, both mouse embryonic stem (ES) cells and mouse induced pluripotent stem (iPS) cells can be directed to the T cell pathway and differentiated into functional, CD8+ T cells specific for the same antigen. However, most efforts to date, including our own has focused on mimicking these niche-specific signals in 2D environments. Based on our promising data in these 2D systems, we hypothesize that a 3D tissue-like microenvironment that can efficiently trigger Delta- Notch and MHC-TcR signaling pathways in cultured ES or iPS cells would provide significant improvement in the efficiency of T cell generation. Our goal here is to engineer such synthetic, 3D T cell niches (artificial thymus-like microenvironment) to direct pluripotent stem cells into therapeutic, antigen-specific T cells. The specific aims of this exploratory research are: Aim 1: To develop and characterize inverse opal hydrogel scaffolds functionalized with ligands essential for T cell differentiation. Aim 2: To efficiently differentiate mouse ES and iPS cells into early T cells using Notch ligand-functionalized inverse opal hydrogel scaffolds. Aim 3: To efficiently generate antigen-specific, functional CD8+ T cells from the early T cell population obtained in Aim 2, using antigen-loaded, MHC-functionalized inverse opal hydrogels and evaluate their antigen specificity and cross-reactivity to self antigens. PUBLIC HEALTH RELEVANCE: The goal of this two year exploratory project is to develop a three dimensional thymus- like microenvironment to generate functional, antigen-specific T cells from mouse embryonic or induced pluripotent stem cells. Our approach is to mimic the thymic niche by creating scaffolds that present specific T cell differentiation signals in an efficient manner. If successful this could lead to adoptive T cell therapy using stem cell derived cells.